Characterization of the cDNA Encoding alphaIIb and beta3 in normal horses and two horses with Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is an inherited, intrinsic platelet defect characterized by a quantitative or qualitative change in the platelet glycoprotein complex IIb-IIIa (integrin alpha(IIb)beta3). The subunits are encoded by separate genes and both subunits must be expressed for a stable complex to form on the platelet surface; therefore, a defect in either gene can result in GT.     

Glanzmann thrombasthenia in a 17-year-old Peruvian Paso mare

A 17-year-old Peruvian Paso mare was evaluated for bilateral epistaxis that had been present for at least 3 years. The mare had mild anemia, platelet count within the reference interval, unremarkable coagulation times, and a negative Coggins test. On endoscopic examination, structural abnormalities were not observed in the nasal cavities, pharynx, larynx, trachea, or either guttural pouch, but petechiation was noted in the nasal mucosa. Additional tests revealed prolonged cutaneous bleeding time, normal concentration of von Willebrand factor antigen, an abnormal clot retraction test, and failure of plalelet aggregation in response to agonists, suggesting a functional disorder of platelets. Genetic analysis indicated the horse was homozygous for a 10-base-pair deletion that included the last 3 base pairs of exon 11 and the first 7 base pairs of intron 11 of the gene encoding glycoprotein IIb. The diagnosis was Glanzmann thrombasthenia (GT) caused by a structural defect in glycoprotein IIb. GT is an autosomal recessive disorder caused by a defect in the glycoprotein IIb-IIIa complex on platelet surfaces. Separate genes encode each glycoprotein, and mutations in either gene can result in GT. This case of GT is unique given the age of the mare at the time of diagnosis. We conclude that GT, although an inherited disorder, should be considered in horses with suspected dysfunctional platelets, regardless of age.     

Glanzmann thrombasthenia in an Oldenbourg filly

An 18-month-old Oldenbourg filly was presented with a bleeding diathesis. Laboratory testing included platelet count, gingival bleeding time, prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor (vWf) antigen, clottable fibrinogen, clot retraction time, PFA-100 closure time, platelet aggregometry (on platelet-rich plasma), and thrombelastography (TEG). TEG was performed by using kaolin and tissue factor as coagulation activators. Expression of the platelet receptor for fibrinogen was assessed by flow cytometry by using anti CD41 (alpha(IIb) or glycoprotein IIb)/CD61 (beta(III) or glycoprotein IIIa) and anti-CD41 antibodies. Abnormal laboratory findings included prolonged oral mucosal bleeding time (>12 hours), prolonged closure time with collagen/ADP (>300 seconds), and absence of clot retraction after 60 minutes. TEG reaction times were similar with kaolin and tissue factor in the patient and a control horse. However, maximum amplitudes in the patient were decreased with both kaolin (43.7 mm; control, 63.9 mm) and tissue factor (37.7 mm; control, 57.8 mm). Platelet aggregation responses to ADP and collagen were profoundly reduced in the affected horse compared with a control. Flow cytometry showed an absence of CD41 and decreased expression of CD41/CD61-reacting antigen on the patient's platelets compared with those from a control horse. The laboratory findings supported a diagnosis of Glanzmann thrombasthenia, likely caused by a mutation in the gene encoding the GPIIb subunit.     

Clinical, biochemical, and molecular aspects of Glanzmann's thrombasthenia in humans and dogs

Glanzmann's thrombasthenia (GT) is an inherited, intrinsic platelet function defect that involves the platelet glycoprotein complex IIb-IIIa, also known as the fibrinogen receptor and the integrin alphaIIbbeta3. The defect was originally described by Dr. Glanzmann in humans in 1918 as a bleeding disorder that differed clinically from other known coagulopathies. Over the decades that followed, researchers determined the biochemical and molecular basis for the disease in humans. Otterhounds with thrombasthenic thrombopathia, described in the 1960s, were the only animal model that closely resembled the disease described in humans until 1996. At that time, a Great Pyrenees dog was identified with unequivocal clinical and biochemical features of Type I GT The cDNA encoding for glycoproteins IIb and IIIa were sequenced in normal dogs in 1999, allowing for identification of specific mutations causing Type I GT in both Otterhounds and Great Pyrenees dogs. Knowing the molecular basis for Type I GT in dogs as well as the cDNA sequences in normal dogs should enhance the understanding of structure/function relationships of the alphaIIbbeta3 integrin and provide an excellent animal model for studies aimed at correction of GT in humans. The following review focuses on the structure and function of this platelet receptor and reviews the molecular, biochemical, and clinical aspects of Glanzmann's thrombasthenia in humans and dogs.     

Effect of a supplement of partly hydrolized straw meal to a diet of fish meal and horse beans on N balance and amino acid excretion in the feces of swine

2 test pigs each (male castrated pigs) of an average live weight of 51-71 kg received either a wheat/fish meal diet (Ia) or a wheat/horse bean diet (IIa). Due to a supplement of partly hydrolysed straw meal the crude fibre content was increased from 3.0% (= Ia) to 10.1% (= Ib) and from 5.2% (= IIa) to 12.1% (= IIb) in the dry matter. This straw meal supplement decreased the apparent digestibility of the crude protein in I from 92.5 to 85.7% and in II from 89.0 to 79.0%. N-excretion in faeces/100 g DM-intake followed the regression line: y = 228 + 31.2x(r = 0.98) y = mg N/100 g DM-intake x = % crude fibre in the DM of the diet The excretion in faeces/100 g DM-intake of 15 investigated amino acids, too, in each case showed a significant increase with the rising crude fibre content of the diet. The widest growth angle (tan alpha) of all the essential amino acids investigated was shown by lysine and those amino acids with branched chains. The conclusion is that in the presence of a fermentable crude fibre source and a good N-supply those 4 amino acids show a suitable bacterial synthesis rate and a low decomposition rate.     

Two genetic defects in alphaIIb are associated with type I Glanzmann's thrombasthenia in a Great Pyrenees dog: a 14-base insertion in exon 13 and a splicing defect of intron 13

Glannzmann's thrombasthenia (GT) is an autosomal recessive bleeding disorder caused by qualitative or quantitative deficiencies of the platelet membrane glycoprotein alphaIIbbeta3. This is the first report of a molecular genetic basis for type I GT in dogs. As previously reported, a thrombasthenic Great Pyrenees dog (dog No. 1) experienced uncontrolled epistaxis despite results of coagulation screening tests, platelet quantitation, and von Willebrand factor quantitation that were within reference ranges. Platelet aggregation was minimal in response to agonists. Flow cytometry, autoradiography, and immunoblot experiments demonstrated either marked reduction or absence of glycoproteins alphaIIb and beta3. In this study, we report the presence of a 14-base insertion in exon 13 and defective splicing of intron 13 in the alphaIIb gene of two thrombasthenic dogs (Nos. 1 and 8). The insertion disrupted the fourth alphaIIb calcium-binding domain, caused a shift in the reading frame and resulted in a premature termination codon. Possible consequences of this mutation include decreased alphaIIb mRNA stability and production of truncated alphaIIb protein that lacks the transmembrane and cytoplasmic domains and a large portion of the extracellular domain. We identified the dam, sire, and three littermates of dog No. 8 as carriers of the alphaIIb mutation. Canine alphaIIb and beta3 genes share significant homology with the genes in human beings, making canine GT an excellent translational model for human GT. A defined molecular basis for canine GT will enhance ongoing gene therapy research and increase the understanding of structure-function relationships of this integrin.     

An Investigation of the Ability of the Glutaraldehyde Test to Distinguish between Acute and Chronic Inflammatory Disease in Horses

The Glutaraldehyde test (GT), a rapid and inexpensive test, has been utilized empirically for many years in bovine practice for diagnosing inflammatory diseases. GT is used primarily to demonstrate increased serum concentrations of fibrinogen and globulin. Glutaraldehyde binds with free amino groups in fibrinogen and immunoglobulin to create a clot in a first degree chemical reaction. The clotting time of the GT estimates the content of proteins produced in response to inflammation. The applicability of GT for diagnosing inflammation in the horse has never been investigated. The objective of this study was to determine the ability of GT to distinguish between acute and chronic inflammatory disease in horses. Thirty-seven horses with suspected inflammatory diseases were evaluated using the GT, history, complete clinical examination and routine blood analysis. GT-times, laboratory results and clinical outcome were compared statistically. Horses that were determined to be acutely affected (based on history, clinical examination and routine blood analysis) tended to have a negative GT (75%). Results of the GT did not correlate with blood fibrinogen concentration. Positive GT also predicted a fatal outcome in 69% of the clinical cases. The results of this trial indicate that GT can be a useful screening test to distinguish between acute and chronic inflammatory disease in horses.     

伊犁马催乳素受体基因的遗传多态性及其与产奶量的关联分析

试验旨在研究伊犁马催乳素受体(prolactin receptor,PRLR)基因多态性及其与产奶量之间的关联性。以60匹伊犁马为研究对象,选取PRLR基因侧翼区的部分片段,采用PCR-SSCP技术检测其遗传多态性,并与伊犁马的日产奶量进行关联分析,研究PRLR基因的多态性与伊犁马产奶性能的关系。结果显示,PRLR基因侧翼区的2个片段均具有多态性,都存在3种基因型:AA、AB和BB,测序结果显示PRLR-P1的碱基突变位置为g.29764513 G>A,该突变为错义突变,导致其编码的氨基酸由组氨酸变成精氨酸,经关联分析发现,该突变对伊犁马日产奶量有显著影响(P<0.05);PRLR-P2的碱基突变位置为g.30106804 C>A,该突变为同义突变,未导致其编码的氨基酸发生改变,经关联分析发现,该突变对伊犁马日产奶量有显著影响(P<0.05)。伊犁马PRLR基因侧翼区的碱基突变与日产奶量之间存在关联性,且可能是影响伊犁马产奶性能的重要位点。     

An investigation of the ability of the glutaraldehyde test to distinguish between acute and chronic inflammatory disease in horses

The glutaraldehyde test (GT), a rapid and inexpensive test, has been utilized empirically for many years in bovine practice for diagnosing inflammatory diseases. GT is used primarily to demonstrate increased serum concentrations of fibrinogen and globulin. Glutaraldehyde binds with free amino groups in fibrinogen and immunoglobulin to create a clot in a first degree chemical reaction. The clotting time of the GT estimates the content of proteins produced in response to inflammation. The applicability of GT for diagnosing inflammation in the horse has never been investigated. The objective of this study was to determine the ability of GT to distinguish between acute and chronic inflammatory disease in horses. Thirty-seven horses with suspected inflammatory diseases were evaluated using the GT, history, complete clinical examination and routine blood analysis. GT-times, laboratory results and clinical outcome were compared statistically. Horses that were determined to be acutely affected (based on history, clinical examination and routine blood analysis) tended to have a negative GT (75%). Results of the GT did not correlate with blood fibrinogen concentration. Positive GT also predicted a fatal outcome in 69% of the clinical cases. The results of this trial indicate that GT can be a useful screening test to distinguish between acute and chronic inflammatory disease in horses.     

Molecular cloning of horse Hsp90 cDNA and its comparative analysis with other vertebrate Hsp90 sequences

Heat shock protein 90 (Hsp90), a molecular chaperone, is ubiquitous and involved in numerous cellular processes. To contribute to the relatively small collection of vertebrate Hsp90 sequences in the gene data bank, we cloned and sequenced horse (Equus caballus) Hsp90 alpha and beta cDNAs. This enabled identification of horse-specific primers for development of a convenient PCR-based method that could monitor horse stress tolerance. We analyzed the sequence data comparatively and phylogenetically with other Hsp90 cDNA sequences, and identified vertebrate-specific and isoform-specific conserved regions to facilitate future molecular investigations of Hsp90 functions. We found 4 highly conserved regions to vertebrate Hsp90 exclusively and 27 amino acids conserved among but differing between Hsp90 alpha and Hsp90 beta sequences. Protein-based phylogenetic trees revealed high conservation between mammal species within Hsp90 alpha and beta clusters. Comparison of nucleotide and amino acid substitution levels suggests that horse Hsp90 beta has undergone strong purifying selection, while rat Hsp90 beta and hamster Hsp90 alpha have been positively selected. Surprisingly, fish Hsp90 alpha genes clearly clustered with Hsp90 beta genes, and no distinct placement of fish Hsp90 alpha protein was found. The Hsp90 alpha isoform is apparently the result of beta gene duplication. Our results highlight the importance of organism- and isoform-specific Hsp90 functional analyses in describing the role of Hsp90 in cells.     

The Association Analysis of Prolactin Receptor Gene Polymorphisms with Milk Yield in Yili Horse

This experiment was designed to analyze association of prolactin receptor (PRLR) gene polymorphism with milk yield of Yili horse.The number of 60 horse were selected as samples, detected genetic polymorphism of PRLR gene using PCR-SSCP technique and sequencing technology, and then analyzed the polymorphism with milk yield of Yili horse.The results showed that there were two polymorphism fragments of PRLR gene flanking region, there were three genotypes:AA, AB and BB.Two novel SNPs (g.29764513 G>A and g.30106804 C>A) were identified of PRLR gene flanking region by sequencing.The g.29764513 G>A SNP caused amino acid variations as p.33His>Arg, while the g.30106804 C>A SNP was synonymous mutation without causing amino acid changes.Statistical results indicated that the g.29764513 G>A and g.30106804 C>A SNPs were significantly associated with daily milk yield in Yili horse (P<0.05).These mutations might be as a crucial DNA genetic markersfor milk production traits selection in Yili horse.     

Molecular cloning and characterization of the alphaX subunit from CD11c/CD18 horse integrin

This work reports the cloning and sequence determination of the horse alpha subunit of the integrin CD11c/CD18, a marker of dendritic cells. A cDNA clone of 4582 base pairs was obtained. It encodes a protein segment of 1086 amino acid residues of the extracellular domain with 10 potential sites of glycosylation, a transmembrane domain of 32 residues and a C-terminal cytoplasmic tail of 24 residues. A phylogenetic analysis of this integrin shows close similarity (83%) with that of Canis familiaris.     

Synthesis and absorption of cysteine from the hindgut of the horse

The extent to which cysteine synthesised by microbes within the hindgut of the horse is incorporated into plasma cysteine was estimated by an isotopic technique in two horses fed four different diets. The results showed that between 1 per cent and 6 per cent of the plasma cysteine was of microbial origin. It is argued that the maximum contribution of microbial cysteine, and presumably other amino acids of microbial origin, to the plasma pool is 12 per cent of the net supply. These data support the hypothesis that microbial amino acid synthesis within the hindgut of the horse does not significantly affect its amino acid status.     

Oxygen affinity responses to 2,3-diphosphoglycerate, and methaemoglobin formation in horse and human haemoglobins.

The oxygen affinities of horse and human haemoglobins were compared in the absence and presence of the allosteric effector 2,3-diphosphoglycerate (2,3-DPG). Horse haemoglobin solutions showed significantly smaller responses to the presence of 2,3-DPG, and this difference may be due to different amino acid substitutions at position NA2(2)beta. Horse haemoglobin solutions from erythrocytes containing different ratios of the two different haemoglobin types showed similar oxygen affinities in the absence and presence of 2,3-DPG. Horse haemoglobins in solution were found to autoxidise to methaemoglobin much more readily than human haemoglobin under the same conditions, and this is an important consideration when measuring the oxygen affinity of horse haemoglobin solutions. This difference could be due to different amino acid residues at position NA2(2)beta.     

Absorption and use of differently structured protein isolates by rats

Differently structured isolated proteins with the same amino acid composition were tested with growing albino rats in an N-balance and absorption experiment. The following items were tested: --isolated horse bean protein --hydrolysed isolated horse bean protein with an amino acid supplement to make up for losses due to hydrolysis --horse bean protein-casein (1:1) fibres --isolated horse bean protein with an amino acid supplement to achieve the same amino acid concentrations as in the horse bean protein-casein fibres. The lowest digestibility and utilisation values were ascertained for the hydrolysed protein. Endogenous N-excretion calculated according to the isotope dilution method was higher than for intact proteins, the transport of the chyme in the digestive tract and the true absorption of N in the small intestine had significantly diminished. In comparison with horse bean protein supplemented with amino acids, the horse bean protein-casein fibres showed decreased protein utilisation and increased endogenous N-excretion. Independent of the protein fed and of the time after feeding a relatively constant endogenous N-quota of approximately 70% could be ascertained in the chyme in the small intestine. In contrast to this, the relation between exogenous and endogenous N determined from the contents of the large intestine proved to be dependent on the intermediary utilisation of the protein fed.     

Definition of the mutation responsible for maple syrup urine disease in Poll Shorthorns and genotyping Poll Shorthorns and Poll Herefords for maple syrup urine disease alleles.

The organisation of the E1alpha subunit of bovine branched-chain alpha-keto acid dehydrogenase gene was established. c DNA was cloned from Poll Shorthorn x Poll Hereford calves affected with Maple Syrup Urine Disease to identify the mutation responsible for the disease in Poll Shorthorns. Clones containing the c DNA sequences inherited from the Poll Shorthorn sire of the affected calves were identified. Paternal clones were sequenced and a cytidine to thymidine transition was found at nucleotide 1380. The mutation is predicted to substitute leucine in place of a highly conserved proline at codon 372. A polymerase chain reaction procedure was developed for detection of the 1380C-->T mutation in genomic DNA. Three Poll Shorthorn parents of affected calves and three affected Poll Shorthorn x Poll Hereford calves were heterozygous and an affected Poll Shorthorn calf was homozygous for this mutation. An improved polymerase chain reaction procedure was also devised to genotype Poll Herefords for the 248C-->T mutation. The procedures will facilitate disease prevention programs and assist in differential diagnosis of conditions in new-born calves that present with a rapid onset of progressive neurological disease and are characterised histologically by 'status spongiosus'. Maple Syrup Urine Disease (MSUD) is an autosomal recessive defect reported in humans (Danner and Elsas 1989), and in Poll Hereford (PH) and Poll Shorthorn (PS) calves (Harper et al 1986, Healy et al 1992). The clinical, biochemical and pathological manifestations of the disease are identical in the two breeds of cattle, and are characterised by the rapid onset of progressive neurological disease, leading to death within a few days of birth. The disease is caused by a deficiency of activity of the mitochondrial enzyme branched-chain alpha-keto acid dehydrogenase (BCKADH). This deficiency leads to elevated concentrations, in blood and tissues, of branched chain alpha-keto acids and their precursors, the branched chain amino acids, valine, leucine and isoleucine. BCKADH consists of four subunits E1alpha, E1beta, E2 and E3 that are encoded by separate genes, and MSUD may result from deficiency of any of the subunits. In PH s, the disease in caused by premature termination of translation, of the E1alpha subunit, that is induced by a cytidine to thymidine transition exon 2 (248C-->T), that converts the glutamine codon -6 to a stop codon (Q-6ST; Zhang et al 1990). We have shown that MSUD -affected PSxPH calves are heterozygous at the PH locus, illustrating molecular heterogeneity exists for bovine MSUD (Healy and Dennis 1994a). The fact that these crossbred calves are affected, indicates the PS, like the PH mutation, resides in the E1alpha subunit.     

P2Y12 receptor gene mutation associated with postoperative hemorrhage in a Greater Swiss Mountain dog

A novel hereditary disorder of platelets was identified across 5 generations of a family of Greater Swiss Mountain dogs. The first dog identified with the mutation bled excessively following routine ovariohysterectomy and required multiple transfusions. Coagulation screening assays, platelet counts, and von Willebrand factor antigen activity were within reference intervals. Flow cytometric studies indicated that platelets from the affected dog expressed normal levels of glycoproteins IIb and IIIa and responded to 2 platelet-activating agents, convulxin and platelet-activating factor, but not to ADP. Based on DNA studies, a 3 base-pair deletion predicted to result in elimination of a serine from the extracellular domain was identified in the gene encoding P2Y12, an ADP receptor protein located on platelet membranes. Flow cytometric analysis of platelets and studies of DNA performed concurrently on 2 unrelated Greater Swiss Mountain dogs were unremarkable. The mutation was subsequently identified in the sire, the maternal grand-dam, a maternal great grandparent, a paternal great grandparent, and a great-great grandparent. The sire was homozygous, but had not yet been identified as having a hemostatic disorder; the other 4 dogs were carriers. This is the first report of a mutation in the gene encoding the ADP receptor P2Y12 in a domestic animal. P2Y12 is the same receptor targeted by ticlopidine and clopidogrel, platelet inhibitors used in lieu of aspirin in people at risk for cardiovascular disease; thus, spontaneous bleeding is not expected unless there are other contributing factors. This disorder is particularly troublesome because spontaneous hemorrhage is absent to mild in affected dogs; however, following routine surgical procedures or trauma, excessive bleeding could occur and have possible fatal consequences.     

绵羊催乳素受体基因外显子10的多态性分析

将催乳素受体(prolactin receptor, PRLR)基因作为绵羊高繁殖力的候选基因,对其外显子10设计2对引物, 采用PCR SSCP 技术检测其在常年发情的湖羊及季节性发情的中国美利奴羊、罗米丽羊和罗米丽×中国美利奴（新疆军垦型）中的单核苷酸多态性。结果表明,引物P1、P2 的扩增片段均有多态性。与已知序列相比,P1扩增片段的AB型在第53 bp处出现A→G突变、在81 bp处出现G→A突变, BB型在该片段第53 bp处发生A→G的突变;对于P2扩增片段, CC、DD、CE和EF型均在第89 bp处发生C→T的突变,导致氨基酸由脯氨酸变为亮氨酸(Pro→Leu),DD型还在146 bp处出现了C→G的突变,此突变导致氨基酸由丙氨酸变为甘氨酸(Ala→Gly);CE型在该片段第132 bp处发生G→A的突变,未导致氨基酸的改变;EF型还在第132 bp处和第167 bp处分别发生了G→A、C→T突变,第167 bp处的突变导致氨基酸由脯氨酸变为亮氨酸(Pro→Leu)。通过卡方独立性检验结果发现,4种绵羊在P1、P2引物扩增片段上各基因型的构成与品种间有极显著差异(P<0.01),说明PRLR基因对绵羊的繁殖性状有一定的影响。